Small, biologically active peptides are frequently produced as larger, usually inactive precursors (preprohormones). Specific proteolytic cleavages liberate various peptides from these precursors. A variety of covalent modifications of the precursor molecule and the smaller peptides occur following synthesis of the preprohormone. One such modification is alpha-amidation, in which the carboxy-terminal carboxylic acid group of a peptide becomes blocked with an alpha-amide group. Approximately half of all known bioactive peptides contain amide groups at their C-termini. In most cases, unblocked versions of these peptides are much less active (on the order of 0.1 to 1%) than their amidated derivatives.
While it is possible to synthesize by chemical means small peptides which contain an amide group at the C-terminal end (alpha-amide), larger alpha-amidated peptides are difficult and expensive to produce. Larger peptides are often produced by expression in bacteria or yeast, but these microbes have not been shown to contain enzymes which can catalyze peptide alpha-amidation of expressed peptides. Thus many peptides produced in cultured microbial cells require the action of PAM to achieve full activity.
PAM activity has been detected in porcine, bovine, human and rat pituitary as well as in other species and tissues, such as frog skin. Some of these PAM enzymes have been purified or partially purified. See, for example, Murthy, et al., Journal of Biological Chemistry, Vol. 261, pp. 1815-1822 (1986) and Mizuno, et al., Biochem. Biophys. Res. Comm., Vol. 137, pp. 984-991 (1986). Prior art purification methods of PAM activity have been directed to soluble enzymes from tissue extracts. This has led to the identification of proteins of approximate molecular weight of 60,000 in porcine pituitaries and molecular weights of 38,000 and 54,000 from bovine pituitaries. However, studies have shown that there is very little PAM protein isolatable from natural sources such as bovine and porcine pituitaries. Thus there is a need in the art to obtain a ready source of PAM enzyme.